1. Field of the Invention
The present invention relates to a newly observed catabolite in a body fluid, particularly urine, its association with malignant conditions in those individuals in which it is present in elevated amounts and methods and materials for its detection.
2. Brief Description of the Prior Art
Numerous methods and markers to determine the presence of cancer cells in mammals, including humans, and to monitor the progress of its treatment have been proposed. Senger, et al, U.S. Pat. No. 4,725,538 discloses observation of a 70,000-74,000 molecular weight protein marker. Burzynski, U.S. Pat. No. 4,444,890 discloses a method for the determination in physiological tissues or fluids of antineoplastons, a group of peptides and amino acid derivatives said to be capable of modulating neoplastic disease. DeFazio et al, U.S. Pat. No. 4,447,545 discloses a method of screening for bladder cancer based on a correlation between the respective ratios of C-reactive protein to total protein in urine and serum and the incidence of bladder cancer. Matsumoto et al., U.S. Pat. No. 4,757,003 discloses a method of cancer detection by immunoassay for certain identified glycolipids which have been found to increase in body fluids with a proliferation of cancer cells. Vold, U.S. Pat. No. 4,665,018 discloses that human cancer can be diagnosed/monitored by measuring the levels of certain modified nucleosides, such as N-[(beta-D-ribofuranosyl)purin-6-ylcarbamoyl]-L-threonine, in urine by a quantitative immunoassay that preferably employs a monoclonal anti-modified nucleoside antibody.
In addition to the above patents, many publications have reported that individuals having diagnosed malignancies are known to excrete high concentrations of certain catabolites, such as p-acetamidobenzoate and pterine derivatives, in their urine. For example, Halpern et al., Pterin-6-aldehyde, a Cancer Cell Catabolite: Identification and Application in Diagnosis and Treatment of Human Cancer, Proc. Nat. Acad. Sci. USA, 74:587-591(1977) report the excretion and identification of pterin-6-aldehyde in the culture media of malignant cells, but not in the media of adult normal cells, embryonic cells and amniotic cells. It was also shown by thin-layer chromatography to be present in the urine of cancer patients (concentrations greater than 300 nmol/ml) but not in normals. Pterin-6-carboxylate and pterin but not pterin-6-aldehyde have previously been found in human urine. Rokos et al., Altered Urinary Excretion of Pteridine in Neoplastic Disease, Clin. Chim. Acta., 105:275-286(1980) reports finding elevated levels of neopterin in 70% of cancer patients studied. Biopterin was less frequently increased and xanthopterin was generally raised when neopterin and/or biopterin excretion was high. They concluded that the pathogenic, diagnostic and therapeutic significance of these changes remains to be established.
Stea et al., Urinary Excretion Levels of Unconjugated Pterins in Cancer Patients and Normal Individuals, Clin. Chim. Acta., 113:231-242(1981) report observing a significant increase in the excretion of xanthopterin, neopterin and pterin and a significant decrease in isoxanthopterin. They also report that biopterin levels were only slightly increased and excretion levels of pterin-6-carboxylic acid and 6-hydroxymethylpterin were about equal in cancer patients and normals. They conclude that excretion patterns of pterins appear to correlate with clinical status and that a definite imbalance in pterin, and possibly folate metabolism, is associated with the presence of malignant diseases.
Rao et al., Elevated Urinary Levels of 6-hydroxymethylpterin during malignancy and liver regeneration: a Simple, Non-invasive test for Cancer Detection, Cancer, 48:1656-1663(1981) report a method for determining urinary 6-hydroxymethylpterin levels. Using this method, healthy human subjects were found to excrete this compound at a mean level of 0.121 ug/ml of urine, while patients with various types of cancer excreted levels ranging from 0.3 to 2.0 ug/ml. The mean excretion level for patients with nonmalignant diseases was 0.134 ug/ml. Trehan et al., Urinary 6-Hydroxymethylpterin Levels Accurately Monitor Response to Chemotherapy in Acute Myeloblastic Leukemia, Cancer, 50:114-117(1982) report measuring urinary 6-hydroxymethylpterin levels as an index of disease status in acute myeloblastic leukemia patients on antileukemic drugs.
In contrast, Hausen et al., Urinary pteridines in Patients Suffering from Cancer, Cancer, 53:1634-1636(1984) reports that the levels determined by Rao et al and Trehan et al were of various pteridines and not exclusively 6-hydroxymethylpterin as reported. Hausen et al also take the position that the urinary component which correlates to the status of malignant disease is neopterin.
Jiang et al., Analysis of Urinary Fluorescent Compounds for Cancer Detection, Chin. Med. J., 98:495-496(1985) report a favorable evaluation of the fluorescence detection method described by Rao et al and Trehan et al which they used with minor modifications.
Tamura et al., Urinary Excretion of Pseudouridine in Patients with Hepatocellular Carcinoma, Cancer, 57:1571-1575 (1986) reports the urinary concentration of pseudouridine in 16 of 23 patients with hepatocellular carcinoma was significantly higher than in patients with cirrhosis of the liver or healthy controls.
Borek, E., Toward a universal tumour marker, Tumour Biol., 5(1):1-14 (1984) and Borek etal., Recent Results in Cancer Research, 84:301-316 (19 ) review work in examining nucleosides as tumor markers. These markers are derivatives of abnormal transfer RNA derivatives and include methylated purine, methylated pyrimidine, pseudouridine, 2-pyridone-5-carboxamide-N'-ribofuranoside and Beta-aminoisobutyric acid. This approach, as well as the others described, requires extensive purification of the sample prior to analysis by HPLC. For other articles regarding nucleosides as tumor markers, see Fischbein, et al., Cancer Detect Prev., 7(4):247-252 (1984); Kaneko, et al., Biochem Biophys Acta, 802(2):169-174 (1984); Sharma, et al., Cancer Detect Prev., 6(1-2):77-85 (1985); Borek, et al., Cancer Detect Prev., 6(1-2):67-71 (1983); Borek, et al. Am. J. Obstet Gynecol., 146(8):906-910 (1983) and other earlier publications by these workers.
Notwithstanding the efforts reported above, such methods as are available are not suitable for routine clinical testing, such as in screening programs. Recent approaches to isolating and identifying tumor markers have required extensive procedures as described above. Also, while some of the methods require complicated specimen processing, others either lack sufficient sensitivity or have a high rate of false positive results reported.